Nucleotide sequences encoding Fasciated EAR3 (FEA3) and methods of use thereof

ABSTRACT

Methods and compositions for modulating shoot apical meristem size are provided. Methods are provided for modulating the expression of fea3 sequence in a host plant or plant cell to modulate agronomic characteristics such as altered size and number of organs, including plant seeds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/612,106, filed on Jun. 2, 2017, which is a continuation applicationof U.S. Ser. No. 14/384,692, filed Sep. 12, 2014, now U.S. Pat. No.9,701,979, which is a National Stage Application of PCT/US2013/030672,filed Mar. 13, 2013, which claims the benefit of U.S. ProvisionalApplication No. 61/610,645, filed Mar. 14, 2012, and U.S. ProvisionalApplication No. 61/751,326, filed Jan. 11, 2013, the entire content ofeach is herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to the field of the genetic manipulation ofplants, particularly the modulation of gene activity and development inplants.

BACKGROUND OF THE INVENTION

Leaves and the axillary meristems that generate branches and flowers areinitiated in regular patterns from the shoot apical meristem (SAM). Thecells of the shoot apical meristem summit serve as stem cells thatdivide to continuously displace daughter cells to the surroundingregions, where they are incorporated into differentiated leaf or flowerprimordia. The meristems are thus capable of regulating their sizeduring development by balancing cell proliferation with theincorporation of cells into new primordia. The SAM provides all aerialparts of plant body. The central concept of stem cells regulation isknown by the signal pathway of CLAVATA/WUSCHEL (CLV/WUS) genes. Loss ofCLV1, CLV2, or CLV3 activity in Arabidopsis causes accumulation ofundifferentiated cells in the shoot apex, indicating that CLV genestogether promote the timely transition of stem cells intodifferentiation pathways, or repress stem cell division, or both(Fletcher et al. (1999) Science 283:1911-1914; Taguchi-Shiobara et al.(2001) Genes and Development 15:2755-5766; Trotochaud et al. (1999)Plant Cell 11:393-405; Merton et al. (1954) Am. J. Bot. 41:726-32;Szymkowiak et al. (1992) Plant Cell 4:1089-100; Yamamoto et al. (2000)Biochim. Biophys. Acta. 1491:333-40). The maize orthologue of CLV1 isTD1 (Bommert et al. (2005) Development 132:1235-1245). The maizeorthologue of CLV2 is FEA2 (Taguchi-Shiobara et al. (2001) Genes Dev. 6515:2755-2766). It is desirable to be able to control the size andappearance of shoot and floral meristems, to give increased yields ofleaves, flowers, and fruit. Accordingly, it is an object of theinvention to provide novel methods and compositions for the modulationof meristem development.

SUMMARY OF THE INVENTION

In one embodiment, the current invention provides a method of producinga transgenic plant with decreased expression of endogenous fea3, themethod comprising the steps of (a) introducing into a regenerable plantcell a recombinant construct comprising a polynucleotide sequenceoperably linked to a promoter, wherein the expression of thepolynucleotide sequence reduces endogenous fea3 expression; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; and (c) selecting a transgenic plant of (b),wherein the transgenic plant comprises the recombinant DNA construct andexhibits a decrease in expression of fea3, when compared to a controlplant not comprising the recombinant DNA construct.

In another embodiment, the current invention provides a method ofproducing a transgenic plant with decreased expression of endogenousfea3, the method comprising the steps of (a) introducing into aregenerable plant cell a recombinant DNA construct comprising anisolated polynucleotide operably linked, in sense or antisenseorientation, to a promoter functional in a plant, wherein thepolynucleotide comprises: (i) the nucleotide sequence of SEQ ID NO:1, 2or 4; (ii) a nucleotide sequence with at least 90% sequence identity,based on the Clustal W method of alignment, when compared to SEQ IDNO:1, 2 or 4; (iii) a nucleotide sequence of at least 100 contiguousnucleotides of SEQ ID NO:1, 2 or 4; (iv) a nucleotide sequence that canhybridize under stringent conditions with the nucleotide sequence of(i); or (v) a modified plant miRNA precursor, wherein the precursor hasbeen modified to replace the miRNA encoding region with a sequencedesigned to produce a miRNA directed to SEQ ID NO:1, 2 or 4; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; and (c) selecting a transgenic plant of (b),wherein the transgenic plant comprises the recombinant DNA construct andexhibits a decrease in expression of fea3, when compared to a controlplant not comprising the recombinant DNA construct.

One embodiment of the invention is a method of producing a transgenicplant with alteration of an agronomic characteristic, the methodcomprising the steps of (a) introducing into a regenerable plant cell arecombinant DNA construct comprising an isolated polynucleotide operablylinked to at least one regulatory sequence, wherein the polynucleotideencodes a fragment or a variant of a polypeptide having an amino acidsequence of at least 80% sequence identity, based on the Clustal Wmethod of alignment, when compared to SEQ ID NO:3 or 5, wherein thefragment or the variant confers a dominant-negative phenotype in theregenerable plant cell; (b) regenerating a transgenic plant from theregenerable plant cell after step (a), wherein the transgenic plantcomprises in its genome the recombinant DNA construct; and (c) selectinga transgenic plant of (b), wherein the transgenic plant comprises therecombinant DNA construct and exhibits an alteration of at least oneagronomic characteristic selected from the group consisting of: earmeristem size, kernel row number, leaf number, inflorescence number,branching within the inflorescence, flower number, fruit number, seednumber, root branching, root biomass, root lodging, biomass and yield,when compared to a control plant not comprising the recombinant DNAconstruct.

Another embodiment of the current invention is the above method whereinexpression of the polypeptide of part (a) in a plant line having thefea3 mutant genotype is capable of partially or fully restoring thewild-type phenotype.

One embodiment of the current invention is a method of identifying aweaker allele of fea3, the method comprising the steps of (a) performinga genetic screen on a population of mutant maize plants (b) identifyingone or more mutant maize plants that exhibit weak fea3 phenotype than afea3 null plant; and (c) identifying the weak fea3 allele from themutant maize plant with weaker fea3 phenotype.

One embodiment of the current invention is a method of identifying aweaker allele of fea3, the method comprising the steps of: (a) geneshuffling using SEQ ID NOS:1, 2 or 4; (b) transforming the shuffledsequences from step (a) into a population of regenerable plant cells;(c) regenerating a population of transformed plants from the populationof transformed regenerable plant cells of step (b); (d) screening thepopulation of transformed plants from step (c) for weak fea3 phenotype;and (e) identifying the weak fea3 allele from the transformed plantexhibiting weak fea3 phenotype.

One embodiment of the invention is a plant in which expression of theendogenous fea3 gene is inhibited relative to a control plant. Anotherembodiment of the current invention is a method of making said plant,the method comprising the steps of (a) introducing a mutation into theendogenous fea3 gene; and (b) detecting the mutation, wherein themutation is effective in inhibiting the expression of the endogenousfea3 gene. In one embodiment, the steps (a) and (b) are done usingTargeting Induced Local Lesions IN Genomics (TILLING) method. Inembodiment, the mutation is a site-specific mutation.

One embodiment of the invention is a plant that exhibits weaker fea3phenotype relative to a wild-type plant. Another embodiment is a methodof making said plant wherein the method comprises the steps of: (a)introducing a transposon into a germ plasm containing an endogenous fea3gene; (b) obtaining progeny of the germplasm of step (a); (c) andidentifying a plant of the progeny of step (b) in which the transposonhas inserted into the endogenous FEA3 gene and a reduction of expressionof fea3 is observed. Step (a) may further comprise introduction of thetransposon into a regenerable plant cell of the germ plasm bytransformation and regeneration of a transgenic plant from theregenerable plant cell, wherein the transgenic plant comprises in itsgenome the transposon.

In one embodiment, the methods described above wherein the methodfurther comprises the steps of (a) introducing into a regenerable plantcell a recombinant construct comprising the weak fea3 allele identifiedby the methods described above; (b) regenerating a transgenic plant fromthe regenerable plant cell after step (a), wherein the transgenic plantcomprises in its genome the recombinant DNA construct; and (c) selectinga transgenic plant of (b), wherein the transgenic plant comprises therecombinant DNA construct and exhibits a weak fea3 phenotype, whencompared to a control plant not comprising the recombinant DNAconstruct.

Another embodiment is a method of producing a transgenic plant with analteration in agronomic characteristic, the method comprising (a)introducing into a regenerable plant cell a recombinant DNA constructcomprising an isolated polynucleotide operably linked, in sense orantisense orientation, to a promoter functional in a plant, wherein thepolynucleotide comprises: (i) the nucleotide sequence of SEQ ID NO:1, 2or 4; (ii) a nucleotide sequence with at least 90% sequence identity,based on the Clustal W method of alignment, when compared to SEQ IDNO:1, 2 or 4; (iii) a nucleotide sequence of at least 100 contiguousnucleotides of SEQ ID NO:1, 2 or 4; (iv) a nucleotide sequence that canhybridize under stringent conditions with the nucleotide sequence of(i); or (v) a modified plant miRNA precursor, wherein the precursor hasbeen modified to replace the miRNA encoding region with a sequencedesigned to produce a miRNA directed to SEQ ID NO:1, 2 or 4; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; and (c) selecting a transgenic plant of (b),wherein the transgenic plant comprises the recombinant DNA construct andexhibits an alteration in at least one agronomic characteristic selectedfrom the group consisting of: enlarged ear meristem, kernel row number,seed number, root branching, root biomass, root lodging, biomass andyield, when compared to a control plant not comprising the recombinantDNA construct. Another embodiment is the plant produced by this method.

One embodiment is a method of expressing a heterologous polynucleotidein a plant, the method comprising (a) transforming a regenerable plantcell with a recombinant DNA construct comprising a heterologouspolynucleotide operably linked to a second polynucleotide, wherein thesecond polynucleotide is a FEA3 promoter (b) regenerating a transgenicplant from the regenerable plant cell after step (a), wherein thetransgenic plant comprises in its genome the recombinant DNA construct;and (c) selecting a transgenic plant of (b), wherein the transgenicplant comprises the recombinant DNA construct and further wherein theheterologous polynucleotide is expressed in the transgenic plant.Another embodiment is the plant comprising in its genome a recombinantDNA construct comprising a heterologous polynucleotide operably linkedto a second polynucleotide, wherein the second polynucleotide is a FEA3promoter and wherein the heterologous polynucleotide is expressed in theplant.

Another embodiment is a method of identifying a first maize plant or afirst maize germplasm that has an alteration of at least one agronomiccharacteristic, the method comprising detecting in the first maize plantor the first maize germplasm at least one polymorphism of a marker locusthat is associated with said phenotype, wherein the marker locus encodesa polypeptide comprising an amino acid sequence selected from the groupconsisting of: a) an amino acid sequence having at least 90% and lessthan 100% sequence identity to SEQ ID NO:3 or 5, wherein expression ofsaid polypeptide in a plant or plant part thereof results in analteration of at least one agronomic characteristic selected from thegroup consisting of: ear meristem size, kernel row number, inflorescencenumber, branching within the inflorescence, flower number, fruit number,and seed number, when compared to a control plant, wherein the controlplant comprises SEQ ID NO:3 or 5. Another embodiment is the above methodwherein said polypeptide comprises the sequence set forth in SEQ IDNO:23, 25 or 27.

The invention includes a recombinant DNA construct comprising anisolated polynucleotide of the current invention operably linked, insense or antisense orientation, to a promoter that is shoot apicalmeristem specific or shoot apical meristem preferred.

This invention includes a vector, cell, plant, or seed comprising any ofthe recombinant DNA constructs described in the present invention.

The invention encompasses plants produced by the methods describedherein.

The invention also encompasses regenerated, mature and fertiletransgenic plants comprising the recombinant DNA constructs describedabove, transgenic seeds produced therefrom, T1 and subsequentgenerations. The transgenic plant cells, tissues, plants, and seeds maycomprise at least one recombinant DNA construct of interest.

In one embodiment, the plant is selected from the group consisting of:Arabidopsis, tomato, maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, sugar cane and switchgrass.

In one embodiment, the plant comprising the recombinant constructsdescribed in the present invention is a monocotyledonous plant. Inanother embodiment, the plant comprising the recombinant constructsdescribed in the present invention is a maize plant.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. § 1.822.

FIG. 1 shows the map-based cloning approach used to isolate thefea3-Reference allele.

FIG. 2A shows RT-PCR data showing the expression analysis of fea2 andfea3 in different tissues. The different tissues analyzed are RAM: rootapical meristem; RE: root elongation zone; RAM(I): RAM of lateral root;SAM; shoot apical meristem (including leaf primordia); EM: earinflorescence meristem FIG. 2B shows fea3 expression in situ. FIG. 2Cshows western blot with anti-RFP antibody of RFP-tagged FEA3, ofmembrane fractionated samples from non-transgenic WT and transgenicplants expressing RFP tagged FEA3 protein. “T” is the “total,unfractionated sample”, “S” is the soluble fraction and “M” is themembrane fraction.

FIG. 3B-3E shows the fasciated fea3 phenotype in ear developmentcompared to that in a wild-type (wt) plant (FIG. 3A).

FIG. 4A-4C show the phenotypic analysis of fea3/fea2 double mutants.

FIG. 4A shows the comparison between the tassels of double mutantscompared to single mutants and wt plants. FIG. 4B shows the spikeletdensity comparison between double mutants, single mutants and wt plants.FIG. 4C shows a comparison between double mutant ear phenotypes comparedto single mutants and wt plants.

FIG. 5A and FIG. 5B shows a comparison between wt plants, fea2 and fea3plants in the CLV3 peptide root assay. FIG. 5B shows the quantitativeanalysis.

FIG. 6 shows a quantitative analysis of the comparison between wtplants, fea2 and fea3 plants in the CLV3-like peptide root assay.

FIG. 7A and FIG. 7B shows wt and fea3 embryos cultured in the presenceof FCP1 and scrambled peptide. FIG. 7A shows wt and fea3 embryo SAMgrowth, and

FIG. 7B shows a quantitative analysis of the same.

FIG. 8A-8C show the phenotypic analysis of fea3/td1 double mutants.

The sequence descriptions (Table 1) and Sequence Listing attached heretocomply with the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. § 1.822.

SEQ ID NO:1 is the nucleotide sequence of the fea3 wt gene.

SEQ ID NO:2 is the coding sequence of wt fea3.

SEQ ID NO:3 is the amino acid sequence of wt fea3.

SEQ ID NO:4 is the coding sequence of alternatively spliced shorterfea3.

SEQ ID NO:5 is the amino acid sequence of alternatively spliced shorterfea3.

SEQ ID NO:6 is the amino acid sequence encoded by the nucleotidesequence corresponding to the locus At1g68780 (Arabidopsis thaliana).

SEQ ID NO:7 is the amino acid sequence encoded by the nucleotidesequence corresponding to the locus At1g13230 (Arabidopsis thaliana).

SEQ ID NO:8 is the amino acid sequence encoded by the nucleotidesequence corresponding to the locus At3g25670 (Arabidopsis thaliana).

SEQ ID NO:9 is the amino acid sequence corresponding to the locusLOC_Os05g43140.1, a rice (japonica) predicted protein from the MichiganState University Rice Genome Annotation Project Osa1 release 6 (January2009).

SEQ ID NO:10 is the amino acid sequence corresponding to Sb03g008380, asorghum (Sorghum bicolor) predicted protein from the Sorghum JGI genomicsequence version 1.4 from the US Department of energy Joint GenomeInstitute.

SEQ ID NO:11 is the amino acid sequence corresponding to Sb03g008360, asorghum (Sorghum bicolor) predicted protein from the Sorghum JGI genomicsequence version 1.4 from the US Department of energy Joint GenomeInstitute.

SEQ ID NO:12 is the amino acid sequence corresponding to Glyma20g32610,a soybean (Glycine max) predicted protein from predicted codingsequences from Soybean JGI Glyma1.01 genomic sequence from the USDepartment of energy Joint Genome Institute.

SEQ ID NO:13 is the amino acid sequence corresponding to Glyma10g34950,a soybean (Glycine max) predicted protein from predicted codingsequences from Soybean JGI Glyma1.01 genomic sequence from the USDepartment of energy Joint Genome Institute.

SEQ ID NO:14 is the amino acid sequence corresponding to Glyma02g11350,a soybean (Glycine max) predicted protein from predicted codingsequences from Soybean JGI Glyma1.01 genomic sequence from the USDepartment of energy Joint Genome Institute.

SEQ ID NO:15 is the amino acid sequence corresponding to Glyma01g22730,a soybean (Glycine max) predicted protein from predicted codingsequences from Soybean JGI Glyma1.01 genomic sequence from the USDepartment of energy Joint Genome Institute.

SEQ ID NO:16 is the amino acid sequence corresponding to Glyma05g07800,a soybean (Glycine max) predicted protein from predicted codingsequences from Soybean JGI Glyma1.01 genomic sequence from the USDepartment of energy Joint Genome Institute.

SEQ ID NO:17 is the amino acid sequence corresponding to Glyma17g13210,a soybean (Glycine max) predicted protein from predicted codingsequences from Soybean JGI Glyma1.01 genomic sequence from the USDepartment of energy Joint Genome Institute.

SEQ ID NO:18 is the nucleotide sequence of a fea3 homolog from Ascelpiassyriaca.

SEQ ID NO:19 is the amino acid sequence encoded by the nucleotidesequence of SEQ ID NO:18.

SEQ ID NO:20 is the nucleotide sequence of fea3-0 reference allele.

SEQ ID NO:21 is the protein sequence of fea3-0 reference allele, encodedby SEQ ID NO:20.

SEQ ID NO:22 is the nucleotide sequence of the EMS mutant fea3-1.

SEQ ID NO:23 is the protein sequence of the EMS mutant allele fea3-1,encoded by the nucleotide sequence given in SEQ ID NO:22.

SEQ ID NO:24 is the nucleotide sequence of the EMS mutant fea3-2.

SEQ ID NO:25 is the protein sequence of the EMS mutant allele fea3-2,encoded by the nucleotide sequence given in SEQ ID NO:24.

SEQ ID NO:26 is the nucleotide sequence of the EMS mutant fea3-3.

SEQ ID NO:27 is the protein sequence of the EMS mutant allele fea3-3,encoded by the nucleotide sequence given in SEQ ID NO:26.

SEQ ID NO:28 is the nucleotide sequence of the FEA3 promoter.

SEQ ID NO:29 is the nucleotide sequence encoding the signal peptide ofthe FEA3 protein.

SEQ ID NO:30 is the nucleotide sequence encoding the RFP-FEA3 fusionprotein.

SEQ ID NO:31 is the nucleotide sequence of the FEA3 3′-UTR.

SEQ ID NOS:32-38 are the sequences of the peptides (ZCL3, FCP1, CLV3,CLE20, CLE40, ZCL21 and ZCL23 respectively) used for the CLV3/CLV3-likepeptide assay described in Example 10.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

The Sequence Listing contains the one letter code for nucleotidesequence characters and the three letter codes for amino acids asdefined in conformity with the IUPAC-IUBMB standards described inNucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(No. 2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. § 1.822.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A monocot of the current invention includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current invention includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

A “trait” refers to a physiological, morphological, biochemical, orphysical characteristic of a plant or particular plant material or cell.In some instances, this characteristic is visible to the human eye, suchas seed or plant size, or can be measured by biochemical techniques,such as detecting the protein, starch, or oil content of seed or leaves,or by observation of a metabolic or physiological process, e.g. bymeasuring tolerance to water deprivation or particular salt or sugarconcentrations, or by the observation of the expression level of a geneor genes, or by agricultural observations such as osmotic stresstolerance or yield.

“Agronomic characteristic” is a measurable parameter including but notlimited to, ear meristem size, tassel size, greenness, yield, growthrate, biomass, fresh weight at maturation, dry weight at maturation,fruit yield, seed yield, total plant nitrogen content, fruit nitrogencontent, seed nitrogen content, nitrogen content in a vegetative tissue,total plant free amino acid content, fruit free amino acid content, seedfree amino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root branching, root biomass, root lodging, harvestindex, stalk lodging, plant height, ear height, ear length, salttolerance, early seedling vigor and seedling emergence under lowtemperature stress.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably to refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Coding region” refers to a polynucleotide sequence that whentranscribed, processed, and/or translated results in the production of apolypeptide sequence.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product have been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” or “regulatory elements” are used interchangeablyand refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include, but are not limited to, promoters, translationleader sequences, introns, and polyadenylation recognition sequences.The terms “regulatory sequence” and “regulatory element” are usedinterchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably to refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in a nullsegregating (or non-transgenic) organism from the same experiment.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant). The term “crossing” refers to the act offusing gametes via pollination to produce progeny.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, a desirable phenotype, e.g., increased cell walldigestibility, or alternatively, is an allele that allows theidentification of plants with decreased cell wall digestibility that canbe removed from a breeding program or planting (“counterselection”). Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype, or alternatively, segregates with the unfavorableplant phenotype, therefore providing the benefit of identifying plants.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The terms“suppression”, “suppressing” and “silencing”, used interchangeablyherein, include lowering, reducing, declining, decreasing, inhibiting,eliminating or preventing. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches.Silencing may be targeted to coding regions or non-coding regions, e.g.,introns, 5′-UTRs and 3′-UTRs, or both.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target isolated nucleic acid fragment(U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA maybe with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target gene or geneproduct. “Sense” RNA refers to RNA transcript that includes the mRNA andcan be translated into protein within a cell or in vitro. Cosuppressionconstructs in plants have been previously designed by focusing onoverexpression of a nucleic acid sequence having homology to a nativemRNA, in the sense orientation, which results in the reduction of allRNA having homology to the overexpressed sequence (see Vaucheret et al.,Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).Cosuppression constructs may contain sequences from coding regions ornon-coding regions, e.g., introns, 5′-UTRs and 3′-UTRs, or both.

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication No.WO 98/36083 published on Aug. 20, 1998).

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., Nature 391:806 (1998)). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., Trends Genet.15:358 (1999)).

Small RNAs play an important role in controlling gene expression.Regulation of many developmental processes, including flowering, iscontrolled by small RNAs. It is now possible to engineer changes in geneexpression of plant genes by using transgenic constructs which producesmall RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA orDNA target sequences. When bound to RNA, small RNAs trigger either RNAcleavage or translational inhibition of the target sequence. When boundto DNA target sequences, it is thought that small RNAs can mediate DNAmethylation of the target sequence. The consequence of these events,regardless of the specific mechanism, is that gene expression isinhibited.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24nucleotides (nt) in length that have been identified in both animals andplants (Lagos-Quintana et al., Science 294:853-858 (2001),Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al.,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001);Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., Genes.Dev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002);Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). They are processedfrom longer precursor transcripts that range in size from approximately70 to 200 nt, and these precursor transcripts have the ability to formstable hairpin structures.

MicroRNAs (miRNAs) appear to regulate target genes by binding tocomplementary sequences located in the transcripts produced by thesegenes. It seems likely that miRNAs can enter at least two pathways oftarget gene regulation: (1) translational inhibition; and (2) RNAcleavage. MicroRNAs entering the RNA cleavage pathway are analogous tothe 21-25 nt short interfering RNAs (siRNAs) generated during RNAinterference (RNAi) in animals and posttranscriptional gene silencing(PTGS) in plants, and likely are incorporated into an RNA-inducedsilencing complex (RISC) that is similar or identical to that seen forRNAi.

The term “locus” generally refers to a genetically defined region of achromosome carrying a gene or, possibly, two or more genes so closelylinked that genetically they behave as a single locus responsible for aphenotype. When used herein with respect to Fea3, the “Fea3 locus” shallrefer to the defined region of the chromosome carrying the Fea3 geneincluding its associated regulatory sequences.

A “gene” shall refer to a specific genetic coding region within a locus,including its associated regulatory sequences. One of ordinary skill inthe art would understand that the associated regulatory sequences willbe within a distance of about 4 kb from the Fea3 coding sequence, withthe promoter located upstream.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leaves, stems, pollen, orcells, that can be cultured into a whole plant.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the Megalign® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal W method of alignment.

The Clustal W method of alignment (described by Higgins and Sharp,CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci.8:189-191 (1992)) can be found in the MegAlign™ v6.1 program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Default parameters for multiple alignment correspond to GAPPENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNATransition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA WeightMatrix=IUB. For pairwise alignments the default parameters areAlignment=Slow-Accurate, Gap Penalty=10.0, Gap Length=0.10, ProteinWeight Matrix=Gonnet 250 and DNA Weight Matrix=IUB.

After alignment of the sequences, using the Clustal W program, it ispossible to obtain “percent identity” and “divergence” values by viewingthe “sequence distances” table on the same program; unless statedotherwise, percent identities and divergences provided and claimedherein were calculated in this manner.

The present invention includes the following isolated polynucleotidesand polypeptides:

An isolated polynucleotide comprising: (i) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal W method of alignment, when compared to SEQ ID NO:3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 23, 25 or 27; or (ii) afull complement of the nucleic acid sequence of (i), wherein the fullcomplement and the nucleic acid sequence of (i) consist of the samenumber of nucleotides and are 100% complementary. Any of the foregoingisolated polynucleotides may be utilized in any recombinant DNAconstructs (including suppression DNA constructs) of the presentinvention. The polypeptide is preferably a FEA3 polypeptide. Thepolypeptide preferably has FEA3 activity.

An isolated polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal W method of alignment, when compared to SEQ ID NO:3, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 21, 23, 25 or 27. Thepolypeptide is preferably a FEA3 polypeptide. The polypeptide preferablyhas FEA3 activity.

An isolated polynucleotide comprising (i) a nucleic acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal W method of alignment, when compared to SEQ IDNO:1, 2, 4, 18, 20, 22, 24 or 26; or (ii) a full complement of thenucleic acid sequence of (i). Any of the foregoing isolatedpolynucleotides may be utilized in any recombinant DNA constructs(including suppression DNA constructs) of the present invention. Thepolypeptide is preferably a FEA3 polypeptide. The polypeptide preferablyhas FEA3 activity.

An isolated polynucleotide comprising a nucleotide sequence, wherein thenucleotide sequence is hybridizable under stringent conditions with aDNA molecule comprising the full complement of SEQ ID NO:1, 2, 4, 18,20, 22, 24 or 26. The polypeptide is preferably a FEA3 polypeptide. Thepolypeptide preferably has FEA3 activity.

An isolated polynucleotide comprising a nucleotide sequence, wherein thenucleotide sequence is derived from SEQ ID NO:1, 2, 4, 18, 20, 22, 24 or26 by alteration of one or more nucleotides by at least one methodselected from the group consisting of: deletion, substitution, additionand insertion. The polypeptide is preferably a FEA3 polypeptide. Thepolypeptide preferably has FEA3 activity.

An isolated polynucleotide comprising a nucleotide sequence, wherein thenucleotide sequence corresponds to an allele of SEQ ID NO:1, 2, 4, 18,20, 22, 24 or 26.

In one embodiment, the present invention includes recombinant DNAconstructs (including suppression DNA constructs). The recombinant DNAconstruct (including suppression DNA constructs) may comprise apolynucleotide of the present invention operably linked, in sense orantisense orientation, to at least one regulatory sequence (e.g., apromoter functional in a plant). The polynucleotide may comprise 100,200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides ofSEQ ID NO:1, 2, 4, 18, 20, 22, 24 or 26. The polynucleotide may encode apolypeptide of the present invention.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

It is well understood by those skilled in the art that differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions.

Promoters that can be used for this invention include, but are notlimited to, shoot apical meristem specific promoters and shoot apicalmeristem preferred promoters. Maize knotted 1 promoter, and promotersfrom genes that are known to be expressed in maize SAM can be used forexpressing the polynucleotides disclosed in the current invention.Examples of such genes include, but are not limited to Zm phabulosa,terminal ear1, rough sheath2, rolled leaf1, zyb14, narrow sheath (Ohtsu,K. et al (2007) Plant Journal 52, 391-404). Promoters from orthologs ofthese genes from other species can be also be used for the currentinvention.

Examples of Arabidopsis promoters from genes with SAM-preferredexpression include, but are not limited to, clv3, aintegumenta-like(ail5, ail6, and ail7) and terminal ear like1, clavata1, wus,shootmeristemless, terminal flower1 (Yadav et al (2009) Proc Natl AcadSci USA. March 24).

PCT Publication Nos. WO 2004/071467 and U.S. Pat. No. 7,129,089 describethe synthesis of multiple promoter/gene/terminator cassette combinationsby ligating individual promoters, genes, and transcription terminatorstogether in unique combinations. Generally, a NotI site flanked by thesuitable promoter is used to clone the desired gene. NotI sites can beadded to a gene of interest using PCR amplification witholigonucleotides designed to introduce NotI sites at the 5′ and 3′ endsof the gene. The resulting PCR product is then digested with NotI andcloned into a suitable promoter/NotI/terminator cassette. Although genecloning into expression cassettes is often done using the NotIrestriction enzyme, one skilled in the art can appreciate that a numberof restriction enzymes can be utilized to achieve the desired cassette.Further, one skilled in the art will appreciate that other cloningtechniques including, but not limited to, PCR-based orrecombination-based techniques can be used to generate suitableexpression cassettes.

In addition, WO 2004/071467 and U.S. Pat. No. 7,129,089 describe thefurther linking together of individual promoter/gene/transcriptionterminator cassettes in unique combinations and orientations, along withsuitable selectable marker cassettes, in order to obtain the desiredphenotypic expression. Although this is done mainly using differentrestriction enzymes sites, one skilled in the art can appreciate that anumber of techniques can be utilized to achieve the desiredpromoter/gene/transcription terminator combination or orientations. Inso doing, any combination and orientation of shoot apicalmeristem-specific promoter/gene/transcription terminator cassettes canbe achieved. One skilled in the art can also appreciate that thesecassettes can be located on individual DNA fragments or on multiplefragments where co-expression of genes is the outcome ofco-transformation of multiple DNA fragments.

The term “root architecture” refers to the arrangement of the differentparts that comprise the root. The terms “root architecture”, “rootstructure”, “root system” or “root system architecture” are usedinterchangeably herein.

As referred to herein, alterations in “Root lodging”, “root branching”and “root biomass” are examples of alterations in “root architecture”.

In general, the first root of a plant that develops from the embryo iscalled the primary root. In most dicots, the primary root is called thetaproot. This main root grows downward and gives rise to branch(lateral) roots. In monocots the primary root of the plant branches,giving rise to a fibrous root system.

The term “altered root architecture” refers to aspects of alterations ofthe different parts that make up the root system at different stages ofits development compared to a reference or control plant. It isunderstood that altered root architecture encompasses alterations in oneor more measurable parameters, including but not limited to, thediameter, length, number, angle or surface of one or more of the rootsystem parts, including but not limited to, the primary root, lateral orbranch root, adventitious root, and root hairs, all of which fall withinthe scope of this invention. These changes can lead to an overallalteration in the area or volume occupied by the root.

One of ordinary skill in the art is familiar with protocols fordetermining alteration in plant root architecture. For example, wt andmutant maize plants can be assayed for changes in root architecture atseedling stage, flowering time or maturity.

Alterations in root architecture can be determined by counting the nodalroot numbers of the top 3 or 4 nodes of the greenhouse grown plants orthe width of the root band.

“Root band” refers to the width of the mat of roots at the bottom of apot at plant maturity. Other measures of alterations in rootarchitecture include, but are not limited to, the number of lateralroots, average root diameter of nodal roots, average root diameter oflateral roots, number and length of root hairs.

The extent of lateral root branching (e.g. lateral root number, lateralroot length) can be determined by sub-sampling a complete root system,imaging with a flat-bed scanner or a digital camera and analyzing withWinRHIZO™ software (Regent Instruments Inc.).

Root lodging is the measure of plants that do not root lodge; plantsthat lean from the vertical axis at an approximately 30 degree angle orgreater would be counted as root lodged.

One can also evaluate alterations in root lodging, root biomass and rootbranching by the ability of the plant to increase yield in field testingwhen compared, under the same conditions, to a control or referenceplant.

Data taken on root phenotype are subjected to statistical analysis,normally a t-test to compare the transgenic roots with that ofnon-transgenic sibling plants. One-way ANOVA may also be used in caseswhere multiple events and/or constructs are involved in the analysis.

One can also evaluate alterations in root lodging, root biomass and rootbranching by the ability of the plant to maintain substantial yield (forexample, at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% yield) in field testing under stress conditions (e.g., nutrientover-abundance or limitation, water over-abundance or limitation,presence of disease), when compared to the yield of a control orreference plant under non-stressed conditions. The wild-type FEA3 or“fasciated ear3” gene encodes a predicted leucine rich repeatreceptor-like protein (LRR-RLP) consisting of 506 amino acids. The terms“wild-type FEA3 gene”, “FEA3 wt gene”, “Fea3 gene” and “FEA3 gene” areused interchangeably herein. Arabidopsis contains three FEA3 orthologuesAt3g25670, At1g 13230, and At1g68780.

LRR-RLPs constitute a large class of LRR-containing proteins (Wang, G.et al (2010) Critical Reviews in Plant Science, 29: 285-299).Structurally, LRR-RLPs can be divided into the following seven distinctdomains: a signal peptide, a cysteine-rich domain, the extracellular LRR(eLRR) domain, a variable domain, an acidic domain, a transmembranedomain, and a short cytoplasmic region (Jones and Jones (1997) Adv. Bot.Res. 24:89-167). The LRR-containing C domain is composed of threesubdomains with a non-LRR island subdomain (C2) that interrupts eLRRsubdomains C1 and C3, although not all RLPs contain a C2 island (Wang,G. et al. (2008) Plant Physiol 147: 503-517).

Our analysis of fea2/fea3 double mutants indicate that fea2 and fea3 actin independent pathways.

Our analysis of td1/fea3 double mutants indicate that td1 and fea3 actin independent pathways.

The term fasciation, from the Latin fascis, meaning bundle, describesvariations in plant form resulting from proliferative growth.

Plants with fea3 mutations, wherein the mutation results in a loss ofFEA3 function or loss of FEA3 expression are also called “fea3 plants”or “fea3 null plants”. “fea3 null plants” exhibit the “fea3 phenotype”or the “fea3 null phenotype”. fea3 plants develop larger meristemsduring inflorescence and floral shoot development, and ear inflorescencemeristems show severe fasciation, suggesting that fea3 normally acts tolimit the growth of these meristems.

Plants with weak fea3 mutations, wherein the mutation results in apartial loss of fea3 function or partial loss of fea3 expression arealso called “fea3 plants with weak fea3 phenotype”. “weak fea3 plants”exhibit the “weak fea3 phenotype”. fea3 plants with weak fea3 allelesexhibit similar phenotype as the fea3 null plants, but to a lesserextent. fea3 plants with weak fea3 alleles may also exhibit partial fea3null phenotype, that is may not exhibit all the fea3 nullcharacteristics. “Weak fea3 alleles” as referred to herein are fea3variants or variants of SEQ ID NOS: 1, 2 or 4, which confer weak fea3phenotype on the plant.

Plants with fea3 mutations that exhibit “null fea3 phenotype” or “weakfea3 phenotype” are referred to herein as plants with “mutant fea3phenotype”.

The term “dominant negative mutation” as used herein refers to amutation that has an altered gene product that acts antagonistically tothe wild-type allele. These mutations usually result in an alteredmolecular function (often inactive) and are characterized by a “dominantnegative” phenotype. A gene variant, a mutated gene or an allele thatconfers “dominant negative phenotype” would confer a “null” or a“mutated” phenotype on the host cell even in the presence of a wild-typeallele.

As used herein, a polypeptide (or polynucleotide) with “FEA3 activity”refers to a polypeptide (or polynucleotide), that when expressed in a“fea3 mutant line” that exhibits the “fea3 mutant phenotype”, is capableof partially or fully rescuing the fea3 mutant phenotype.

The terms “gene shuffling” and “directed evolution” are usedinterchangeably herein. The method of “gene shuffling” consists ofiterations of DNA shuffling followed by appropriate screening and/orselection to generate variants of FEA3 nucleic acids or portions thereofhaving a modified biological activity (Castle et al., (2004) Science304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).

“TILLING” or “Targeting Induced Local Lesions IN Genomics” refers to amutagenesis technology useful to generate and/or identify, and toeventually isolate mutagenised variants of a particular nucleic acidwith modulated expression and/or activity (McCallum et al., (2000),Plant Physiology 123:439-442; McCallum et al., (2000) NatureBiotechnology 18:455-457; and, Colbert et al., (2001) Plant Physiology126:480-484).

TILLING combines high density point mutations with rapid sensitivedetection of the mutations. Typically, ethylmethanesulfonate (EMS) isused to mutagenize plant seed. EMS alkylates guanine, which typicallyleads to mispairing. For example, seeds are soaked in an about 10-20 mMsolution of EMS for about 10 to 20 hours; the seeds are washed and thensown. The plants of this generation are known as M1. M1 plants are thenself-fertilized. Mutations that are present in cells that form thereproductive tissues are inherited by the next generation (M2).Typically, M2 plants are screened for mutation in the desired geneand/or for specific phenotypes.

TILLING also allows selection of plants carrying mutant variants. Thesemutant variants may exhibit modified expression, either in strength orin location or in timing (if the mutations affect the promoter forexample). These mutant variants may even exhibit lower FEA3 activitythan that exhibited by the gene in its natural form. TILLING combineshigh-density mutagenesis with high-throughput screening methods. Thesteps typically followed in TILLING are: (a) EMS mutagenesis (Redei G Pand Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua NH, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82;Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds,Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater,J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press,Totowa, N.J., pp 91-104); (b) DNA preparation and pooling ofindividuals; (c) PCR amplification of a region of interest; (d)denaturation and annealing to allow formation of heteroduplexes; (e)DHPLC, where the presence of a heteroduplex in a pool is detected as anextra peak in the chromatogram; (f) identification of the mutantindividual; and (g) sequencing of the mutant PCR product. Methods forTILLING are well known in the art (U.S. Pat. No. 8,071,840).

Other mutagenic methods can also be employed to introduce mutations inthe FEA3 gene. Methods for introducing genetic mutations into plantgenes and selecting plants with desired traits are well known. Forinstance, seeds or other plant material can be treated with a mutagenicchemical substance, according to standard techniques. Such chemicalsubstances include, but are not limited to, the following: diethylsulfate, ethylene imine, and N-nitroso-N-ethylurea. Alternatively,ionizing radiation from sources such as X-rays or gamma rays can beused.

Other detection methods for detecting mutations in the FEA3 gene can beemployed, e.g., capillary electrophoresis (e.g., constant denaturantcapillary electrophoresis and single-stranded conformationalpolymorphism). In another example, heteroduplexes can be detected byusing mismatch repair enzymology (e.g., CELI endonuclease from celery).CELI recognizes a mismatch and cleaves exactly at the 3′ side of themismatch. The precise base position of the mismatch can be determined bycutting with the mismatch repair enzyme followed by, e.g., denaturinggel electrophoresis. See, e.g., Oleykowski et al., (1998) “Mutationdetection using a novel plant endonuclease” Nucleic Acid Res.26:4597-4602; and, Colbert et al., (2001) “High-Throughput Screening forInduced Point Mutations” Plant Physiology 126:480-484.

The plant containing the mutated fea3 gene can be crossed with otherplants to introduce the mutation into another plant. This can be doneusing standard breeding techniques.

Homologous recombination allows introduction in a genome of a selectednucleic acid at a defined selected position. Homologous recombinationhas been demonstrated in plants. See, e.g., Puchta et al. (1994),Experientia 50: 277-284; Swoboda et al. (1994), EMBO J. 13: 484-489;Offringa et al. (1993), Proc. Natl. Acad. Sci. USA 90: 7346-7350; Kempinet al. (1997) Nature 389:802-803; and, Terada et al., (2002) NatureBiotechnology, 20(10):1030-1034).

Methods for performing homologous recombination in plants have beendescribed not only for model plants (Offringa et al. (1990) EMBO J.October; 9(10):3077-84) but also for crop plants, for example rice(Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S. Nat Biotechnol. 2002;Iida and Terada: Curr Opin Biotechnol. 2004 April; 15(2):1328). Thenucleic acid to be targeted (which may be FEA3 nucleic acid or a variantthereof as hereinbefore defined) need not be targeted to the locus ofFEA3 gene respectively, but may be introduced in, for example, regionsof high expression. The nucleic acid to be targeted may be weak fea3allele or a dominant negative allele used to replace the endogenous geneor may be introduced in addition to the endogenous gene.

Transposable elements can be categorized into two broad classes based ontheir mode of transposition. These are designated Class I and Class II;both have applications as mutagens and as delivery vectors. Class Itransposable elements transpose by an RNA intermediate and use reversetranscriptases, i.e., they are retroelements. There are at least threetypes of Class I transposable elements, e.g., retrotransposons,retroposons, SINE-like elements. Retrotransposons typically containLTRs, and genes encoding viral coat proteins (gag) and reversetranscriptase, RnaseH, integrase and polymerase (pol) genes. Numerousretrotransposons have been described in plant species. Suchretrotransposons mobilize and translocate via a RNA intermediate in areaction catalyzed by reverse transcriptase and RNase H encoded by thetransposon. Examples fall into the Tyl-copia and Ty3-gypsy groups aswell as into the SINE-like and LINE-like classifications (Kumar andBennetzen (1999) Annual Review of Genetics 33:479). In addition, DNAtransposable elements such as Ac, TamI and En/Spm are also found in awide variety of plant species, and can be utilized in the invention.Transposons (and IS elements) are common tools for introducing mutationsin plant cells.

The shoot apical meristem (SAM) regulates its size during development bybalancing stem cell proliferation and the incorporation of daughtercells into primordia. Several “fasciated” mutants with enlargedmeristems have been identified in maize, and can be used to study thegenetic basis of meristem size regulation. Two maize genes, thick tasseldwarf1 (td1; Bommert et al. (2005) Development 132:1235-1245) andfasciated ear2 (fea2; Taguchi-Shiobara et al. (2001) Genes Dev. 6515:2755-2766), are homologous to the Arabidopsis leucine-rich-repeat(LRR) receptor-genes CLAVATA1 (CLV1) and CLAVATA2 (CLV2), respectively.CLV1 and CLV2 were predicted to form a receptor complex that isactivated by the CLV3 ligand and represses the stem cell promotingtranscription factor WUSCHEL. Analysis of fea2/td1 double mutantshowever suggested, that the basic CLV1-CLV2 co-receptor model is likelymore complex, as the fea2/td1 double mutant showed a more severephenotype than either single mutant. Recent analysis in Arabidopsisrevealed that the separate action of three major receptor complexes(CLV1-BAM1 (BARELY ANY MERISTEM1), CLV2-CRN (CORYNE), and RPK2/TOAD2(RECEPTOR-LIKE PROTEIN KINASE2/TOADTOOL2)) is necessary for propermeristem size control in Arabidopsis.

Here we present a phenotypic and molecular characterization of the maizemutant fea3 that causes the over-proliferation of the inflorescencemeristem, leading to enlarged or fasciated meristems. We cloned the fea3gene using a map-based cloning approach and the mutant results from aninsertion of a partial retrotransposon into an exon of the fea3 locus.We confirmed this identity by isolation of three additional alleles offea3 derived from a targeted EMS mutagenesis. The FEA3 gene encodes apredicted leucine rich repeat receptor-like protein, related to fea2.In-situ hybridization and Red Fluorescent Protein-tagged transgenicplants show that FEA3 is expressed in the organizing center of SAM andis also expressed in the root apical meristem. FEA3 is localized in theplasma membrane. To determine if FEA3 responds to a CLV3-related (CLE)peptide, we tested its sensitivity to different peptides. The fea3mutants showed reduced peptide sensitivity, but interestingly theyresponded to a different CLE peptide compared to FEA2. Double mutants offea2/fea3 and td1/fea3 have additive and synergistic fasciatedphenotypes in ear and tassel, indicating that they act in independentpathways that converge on the same downstream target to control meristemsize. Consequently, the function of FEA3 as a receptor protein is in anew pathway distinct from that of TD1 and FEA2.

EMBODIMENTS

In one embodiment, the fea3 variant that can be used in the methods ofthe current invention is one or more of the following fea3 nucleic acidvariants: (i) a portion of a fea3 nucleic acid sequence (SEQ ID NO:1, 2or 4); (ii) a nucleic acid sequence capable of hybridizing with a fea3nucleic acid sequence (SEQ ID NO:1, 2 or 4); (iii) a splice variant of afea3 nucleic acid sequence (SEQ ID NO:1, 2 or 4); (iv) a naturallyoccurring allelic variant of a fea3 nucleic acid sequence (SEQ ID NO:1,2 or 4); (v) a fea3 nucleic acid sequence obtained by gene shuffling;(vi) a fea3 nucleic acid sequence obtained by site-directed mutagenesis;(vii) a fea3 variant obtained and identified by the method of TILLING.

In one embodiment, the levels of endogenous FEA3 expression can bedecreased in a plant cell by antisense constructs, sense constructs, RNAsilencing constructs, RNA interference, artificial microRNAs and genomicdisruptions. Examples of genomic disruption include, but are not limitedto, disruptions induced by transposons, tilling, homologousrecombination.

In one embodiment, a modified plant miRNA precursor may be used, whereinthe precursor has been modified to replace the miRNA encoding regionwith a sequence designed to produce a miRNA directed to FEA3. Theprecursor is also modified in the star strand sequence to correspond tochanges in the miRNA encoding region.

In one embodiment, a nucleic acid variant of FEA3 useful in the methodsof the invention is a nucleic acid variant obtained by gene shuffling.

In one embodiment, a genetic modification may also be introduced in thelocus of a maize FEA3 gene using the technique of TILLING (TargetedInduced Local Lesions In Genomes).

In one embodiment, site-directed mutagenesis may be used to generatevariants of fea3 nucleic acids. Several methods are available to achievesite-directed mutagenesis; the most common being PCR based methods (U.S.Pat. No. 7,956,240).

In one embodiment homologous recombination can also be used toinactivate, or reduce the expression of endogenous FEA3 gene in a plant.

Homologous recombination can be used to induce targeted genemodifications by specifically targeting the FEA3 gene in vivo. Mutationsin selected portions of the FEA3 gene sequence (including 5′ upstream,3′ downstream, and intragenic regions) such as those provided herein aremade in vitro and introduced into the desired plant using standardtechniques. Homologous recombination between the introduced mutated fea3gene and the target endogenous FEA3 gene would lead to targetedreplacement of the wild-type gene in transgenic plants, resulting insuppression of FEA3 expression or activity.

In one embodiment, catalytic RNA molecules or ribozymes can also be usedto inhibit expression of FEA3 gene. It is possible to design ribozymesthat specifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules. The inclusion of ribozyme sequences within antisenseRNAs confers RNA-cleaving activity upon them, thereby increasing theactivity of the constructs. A number of classes of ribozymes have beenidentified. For example, one class of ribozymes is derived from a numberof small circular RNAs that are capable of self-cleavage and replicationin plants. The RNAs can replicate either alone (viroid RNAs) or with ahelper virus (satellite RNAs). Examples of RNAs include RNAs fromavocado sunblotch viroid and the satellite RNAs from tobacco ringspotvirus, lucerne transient streak virus, velvet tobacco mottle virus,Solanum nodiflorum mottle virus and subterranean clover mottle virus.The design and use of target RNA-specific ribozymes has been described.See, e.g., Haseloff et al. (1988) Nature, 334:585-591.

Another method to inactivate the FEA3 gene is by inhibiting expressionis by sense suppression. Introduction of expression cassettes in which anucleic acid is configured in the sense orientation with respect to thepromoter has been shown to be an effective means by which to block thetranscription of a desired target gene. (Napoli et al. (1990), The PlantCell 2:279-289, and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184).

In one embodiment, the FEA3 gene can also be inactivated by, e.g.,transposon based gene inactivation.

In one embodiment, the inactivating step comprises producing one or moremutations in the FEA3 gene sequence, where the one or more mutations inthe FEA3 gene sequence comprise one or more transposon insertions,thereby inactivating the FEA3 gene compared to a corresponding controlplant. For example, the mutation may comprise a homozygous disruption inthe FEA3 gene or the one or more mutations comprise a heterozygousdisruption in the FEA3 gene.

These mobile genetic elements are delivered to cells, e.g., through asexual cross, transposition is selected for and the resulting insertionmutants are screened, e.g., for a phenotype of interest. Plantscomprising disrupted fea3 genes can be crossed with a wt plant. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed. The location of a TN (transposon) within a genomeof an isolated or recombinant plant can be determined by known methods,e.g., sequencing of flanking regions as described herein. For example, aPCR reaction from the plant can be used to amplify the sequence, whichcan then be diagnostically sequenced to confirm its origin. Optionally,the insertion mutants are screened for a desired phenotype, such as theinhibition of expression or activity of fea3 or alteration of anagronomic characteristic.

EXAMPLES

The present invention is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Furthermore, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Cloning of Maize Fea3 Gene

A map-based cloning approach was used to isolate the fea3-0 Referenceallele (SEQ ID NO:20), which was originally mapped on chromosome 3 (FIG.1 ). A partial retrotransposon insertion within a gene encoding aleucine-rich-repeat receptor like protein was identified by finemapping. To confirm that this insertion was the causative mutation, atargeted EMS screen was performed, which allowed us to identify threeadditional alleles of fea3, designated fea3-1, -2 and -3 (SEQ ID NOS:22, 24, and 26 respectively).

fea3 was initially mapped using bulked segregant mapping. A mappingpopulation of 947 individuals was used to place the locus between theBACs c0267MO3 and c0566I18, a region of ˜6 BACs containing ˜25 predictedgenes. Sequencing and expression analysis revealed one candidate, an LRRreceptor like protein that had a small insertion in the fea3-0 allele.Three additional alleles were identified using a targeted EMS screenfrom ˜10,000 M1 plants. Sequencing of each allele revealed an amino acidchange relative to the progenitor, confirming that the correct gene wasisolated.

Example 2 Expression Analysis of FEA2 and FEA3 Genes

RT-PCR was done for FEA2 and FEA3 in different tissues. FIG. 2A showsthe expression of FEA22 and FEA3 in different tissues. FEA2 and FEA3show the strongest expression the shoot apical meristem. FIG. 2B showsthe FEA3 expression in situ, showing expression is detected organizingcenter of meristem. This region overlaps with WUS expression region.This pattern is quite different with other known fasciated ear mutant(Inflorescence transition stage).

Example 3 Maize Mutant Fea3 Phenotype

During vegetative development fea3 mutant plants appear normal. Aftertransition to flowering, however, during early inflorescencedevelopment, fea3 mutants ears (FIG. 3B) show a flattened and enlargedinflorescence meristem (IM) compared to wild type (FIG. 3A). At laterstages of development enlargement of the IM causes fasciation in themutant (FIG. 3C). At maturity wild type ears show regularly spaced andorganized kernel rows (FIG. 3D), whereas fea3 mutant ears show aprogressive enlargement of the ear tip, extra kernel rows and an overallirregular arrangement of rows (FIG. 3E).

Example 4 Fea3/Fea2 Double Mutant Analysis

The tassels of maize fea3/fea2 double mutants are thicker and shortercompared to single mutants (FIG. 4A). Spikelet density was analyzed bycounting spikelets per cm along the main rachis. Double mutants show asignificant increase in spikelet density, indicating additive effectsbetween fea2 and fea3 (FIG. 4B). Similarly, double mutant ear phenotypesshow additive fasciation (FIG. 4C). These results suggest that FEA2 andFEA3 act in different pathways.

Example 5 Clavata3 Peptide Root Assay

In Arabidopsis, CLAVATA2 activity can be detected by responses of rootgrowth to CLAVATA3 (CLV3) peptide. To analyze whether FEA3 and FEA2respond to CLV3, and determine if they act in a common pathway a CLV3peptide assay was performed. B73 and homozygous fea2 and fea3 mutantseedlings were germinated and grown on agar plates containing CLV3peptide. As a control, seedlings were also grown on plates containing amutated version of the peptide and on plates without any peptide. After7 days the length of the primary root was measured. B73 wild type plantsshow strong root growth inhibition as result of response to CLV3peptide, but fea2 mutants do not respond to CLV3 peptide. Interestingly,fea3 mutants respond to CLV3 peptide, even though FEA3 is expressed inroot (FIG. 5 ).

Example 6 Expression of Red Fluorescent Protein from the FEA3 Promoter

A recombinant DNA construct was made to allow for in vivo localizationof FEA3 that has been tagged with Red Fluorescent Protein (RFP). Theconstruct contained the following elements in the 5′ to 3′orientation: 1) FEA3 Promoter; 2) FEA3 signal peptide coding region; 3)RFP-FEA3 fusion protein coding region; and 4) FEA3 3′-UTR. Transgenicmaize plants containing this recombinant DNA construct were produced.Analysis of the transgenic plants revealed that RFP-FEA3 fusion proteinwas expressed in the inflorescence meristem central zone of both the earand the tassel.

To see whether FEA3 is localized in the membrane or the solublefraction, western blot was performed after membrane fractionation.Tissue used was young tassel (about 0.5-3 cm tassel) from the transgenicplant expressing RFP tagged FEA3 protein, as described above. FIG. 2Cshows that RFP tagged FEA3 is localized in the plasma membrane, with thearrow indicating band size of about 83 kD which is expected fusion sizeof RFP tagged FEA3.

Example 7 Clavata3-Like Peptide Root Assay

To analyze whether FEA3 responds to CLV3-like peptides, and determine ifthey act in a common pathway, a CLV3 peptide assay was performed. Thepeptides used were ZCL3 (Zea mays CLE-like 3; SEQ ID NO:32), FCP1 (SEQID NO:33), CLV3 (SEQ ID NO:34), CLE20 (SEQ ID NO:35), CLE40 (SEQ IDNO:36), ZCL21 (Zea mays CLE-like 3; SEQ ID NO:37), and ZCL23 (Zea maysCLE-like 23; SEQ ID NO:38).

The ZCL peptides were found in maize sequences in the NCBI database byhomology search using the CLV3, CLE, and rice related peptides (Fiers etal Plant Cell (2005), 17: 2542-2553; Suzaki et al (2008), Plant Cell,20: 2049-2058).

B73 and fea3 mutant seedlings were germinated and grown on agar platescontaining each 5 μM or 10 μM peptide. As a control, seedlings were alsogrown on plates containing a scramble of the peptide. After 7 days thelength of the primary root was measured. B73 wild type plants showstrong root growth inhibition as result of response to ZCL3 (SEQ IDNO:32), FCP1 (SEQ ID NO:33) and CLV3 (SEQ ID NO:34) peptides.Interestingly, fea3 mutants show less sensitivity to FCP1 peptide (FIG.6 ).

Example 8 Embryo Culture Assay in Presence of FCP1 Peptide

Wt and fea3 embryos were cultured in the presence of 20 μM FCP1 peptide(SEQ ID NO:33) or 20 μM scrambled peptide. For measurement of embryo SAMgrowth, about 10 days after pollination, embryos were sterilized (wholecorn was sterilized, not individual young seeds) and dissected embryosand put the embryos down on the media and the measurement of SAM sizewas done two weeks after planting (embryo culture). WT embryo SAM growthwas found to be strongly inhibited by FCP1, but fea3 embryos showedresistance (FIG. 7A shows an image comparing wt and fea3 embryo SAMgrowth, and FIG. 7B shows a quantitative analysis of the same). For thehistogram shown in FIG. 7B, p<0.0001

Example 9 Fea3/td1 Double Mutant Analysis

The tassels of maize fea3/td1 double mutants are thicker and shortercompared to single mutants (FIG. 4A). Spikelet density was analyzed bycounting spikelets per cm along the main rachis. Double mutants show asignificant increase in spikelet density, indicating additive effectsbetween fea2 and fea3 (FIG. 4B). Similarly, double mutant ear phenotypesshow additive fasciation (FIG. 4C). These results suggest that FEA2 andFEA3 act in different pathways.

Example 10 Analysis of Fea3 Orthologs in Other Plant Species

Arabidopsis, rice, sorghum and soy orthologs of FEA3 can also beanalyzed by doing experiments described in Examples 1-9 for maize FEA3.

What is claimed is:
 1. A modified maize plant comprising an introducedgenetic modification in an endogenous fea3 gene encoding a FEA3polypeptide, wherein the FEA3 polypeptide comprises an amino acidsequence that is at least 95% identical to SEQ ID NO: 3, wherein themodified maize plant comprises an amino acid substitution in the FEA3polypeptide and exhibits increased kernel row number or kernel number,compared to a control maize plant not comprising the geneticmodification.
 2. The maize plant of claim 1, wherein the maize plantexhibits increased kernel row number.
 3. The maize plant of claim 1,wherein the maize plant exhibits increased ear size.
 4. The maize plantof claim 1, wherein the maize plant exhibits reduced tassels.